As genetic technologies continue to advance, scientists around the world maintain their pursuit of higher research endeavors and what they are individually focused on. Tied to this is the need for ever more sequencing and production of genes, of loci, of entire genomes. We have better sequencing machinery now, after all, they may as well be put to use. However, at the same time, there is significant room for improvement in both avenues. Sequencing is one half of the puzzle, but the ability to reproduce desired genes from scratch, perhaps even build entire new genomes as some have already done, is another topic entirely.

The Process of Synthesis

Once a scientist has had a gene sequenced, they are usually going to want a pure sample of it so they can test its properties and put it to use. Isolating the gene from an organism is still the primary method, but often point mutations or other tweaks to the genes are the subject being tested. Depending on the length desired, it is often nowadays more efficient to merely synthesize the gene in whole, without dealing with extraneous genetic material and mutagenic compounds to get the same outcome. This alternative can come in the form of very short polynucleotides which are called oligonucleotides that are made in parallel and then stuck together or, more rarely, full gene and chromosome constructs.

The cost-effective option is to use a massive amount of oligonucleotides, which in turn has become a staple of modern biology. Even so, the method of accomplishing this is quite old in regards to the field of biotechnology. The nucleoside phosphoramidite method was developed over 35 years ago in 1981 by a group of scientists headed by Marvin Caruthers. Since regular nucleotides aren’t reactive enough to chain them together into a strand of genetic material, the nucleoside version without the phosphate group is taken and reacted with phosphoramidite in order to make a molecule that is much more amenable to chaining together. And, thus, oligonucleotides are made in this manner.

But even after subtle improvements and variations on this process over that time frame to now, the scientific community has still only managed to increase the oligonucleotide synthesis amount to around 2 to 3 hundred nucleotides. Far below what is desired or needed for future experiments. Add to this that these longer synthesized products are highly difficult to manufacture, are prone to outright failure, and also can’t be used with all the kinds of sequences that are required and you are left with a branch of biochemistry that is sorely wanting some new revelation.

Finding Other Options

There have certainly been attempts to produce additional techniques, far too many to count even. One of the strongest subfields in this regard is the use of natural enzymes for construction of genetic sequences as is provided to them. There are obvious benefits to this choice, as a water solution rather than a hazardous one can be utilized, the specificity of enzymatic interactions allow for the creation of even longer oligonucleotides, and natural DNA sequences can even be added to or used as a template to start. The ability to fine-tune with such a system would be currently unparalleled.

Researchers at UC Berkeley were determined to be the ones that were successful at creating just such a thing. They decided that the best enzyme among those available for their purposes was terminal deoxynucleotidyl transferase (TdT), which is known to add deoxynucleotide triphosphates (dNTPs), the primary building blocks of nucleic acids, haphazardly to the end of single stranded DNA. This would make it perfect for chaining together nucleotides into a long oligonucleotide. But no one has managed to get TdT to work this way in practice thus far, even though plans of how to make it work have been suggested left and right since practically the beginning in 1985.

The main problem with these plans is that they relied on reverse terminator dNTPs, which are used to be able to control when and which nucleotides are added, so they can block elongation after one nucleotide is added before adding the next. The issue is that these don’t work all that well with TdT. Thus, no one has managed to get either to fit together in all the years since.

Problems Left To Correct

The Berkeley scientists came up with a workaround. What if they took the reverse terminators and attached a labeled NTP that can then be cleaved in a second step to allow TdT to add the next one? It would be a repeatable and controllable two step process. Once tested, it worked perfectly, letting them place the nucleotides one by one as was necessary. The best part is that the NTPs can be linked in different places on the reverse terminators, allowing the design to be general and applicable to multiple forms of experiments.

There are several challenges left to tackle, the authors admit. First off, the current setup is only at a 98% accuracy in regards to which nucleotides are added and they want to bump this up to over 99% and preferably 99.9% before it can be considered useful for the broader scientific world.

They hope to find out why ~1% of the time the reverse terminator doesn’t stop another piece from being incorporated right away. Some of the steps they tested had the accuracy drop to around 93% and they have yet to find out why such a discrepancy occurs with some linkages, so they will be researching that more. Timing of the linking is hypothesized to play a role.

Changing The Future

Overall, this technique could increase the average size of produced oligonucleotides by 5 to 10 times, reducing both time and cost equally. If the researchers are able to bump up their accuracy that small amount they want, then they will have found a way to completely change the genetic synthesis field overnight. The effects of this would ripple throughout all of science and especially for those that rely on creation of gene samples for their research.

The precise outcome of such a triumph would be hard to overstate and the authors don’t oversell it when they say it will prove a revolution for biotechnology, medicine, general research, and beyond.

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Photo CCs: 3D-SIM-3 Prophase 3 color from Wikimedia Commons

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